PROJECT SUMMARY/ABSTRACT The unregulated opening of hemichannels (HCs) formed by the gap junction (GJ) protein Cx43 during myocardial infarction (MI) contributes injury spread, cardiac muscle loss and the formation of an arrhythmic substrate. In the previous period, we demonstrated that the Cx43 mimetic peptide ?CT1 spares left ventricular muscle and contractile function in a mouse ischemia reperfusion (I/R) injury model and determined that the molecular mechanism of this 25 amino acid peptide?s cardioprotective activity was via binding to the H2 domain of Cx43. This interaction induces a cardioprotective Protein Kinase C phosphorylation of Cx43 at serine 368 (S368) ? a post-translational modification known to reduce the activity of HCs. It was further shown that the main niche for HCs ? the GJ perinexus ? demonstrates high concentrations of Nav1.5/Scn5a voltage-gated sodium channels (VGSCs) and that the VGSC beta subunit ?1/Scn1b maintains perinexal adhesion, enabling formation of trans-activating sodium channels that have key assignments in cardiac conduction and arrhythmia. The previous funding period also included completion and publication of results from two Phase II clinical trials on ?CT1. In new preliminary data, we show that a short (9 amino acid) variant of ?CT1, called ?CT11: (1) Potently reduces ventricular infarct size by 48 % in an in vivo mouse model of MI, with significant preservation of echocardiographically-assessed contractile function when given post-ischemia; and (2) Preserves conduction and inhibits transition to discordant alternans in an ex vivo ischemia model. We have reported that like ?CT1, the ?CT11 mechanism is via Cx43 H2 binding and S368 phosphorylation. However, unlike ?CT1 (which incorporates an antennapedia penetration sequence) our data indicates that the ?CT11 reaches it cytoplasmic target, the Cx43 H2 domain, via permeating HCs. During the next funding period our aims are to: (1) Undertake rigorous testing of post-MI ?CT11 efficacy in preserving cardiac muscle and preventing arrhythmias in vivo; (2) Determine whether ?CT11?s mode-of-action involves the short peptide permeating HCs; and (3) Test a novel approach to loading of exosomes with ?CT11 and undertake proof-of-principle testing of this formulation in preclinical models of MI. The significance of these aims are underpinned by: (1) ?CT1 now being in Phase III testing on more than 500 patients; (2) That there is no clinical approach for cardioprotection post-MI; and (3) The growing evidence that Cx43 HC activation is key to other ischemia-related pathologies, including diabetic foot ulcers and cerebral stroke. The studies proposed herein will provide mechanistic understanding and lay the basis for translation of new drugs targeting HCs. It is our premise that this work is a necessary prelude to clinical testing of HC-targeting drugs in humans as muscle-sparing and anti-arrhythmic therapies in the critical hours following a heart attack.